zmmunology/oday, August 1980
30
The role of monoclonal antibody technology in immunop ara sit ol ogy D. S. R o w e World Health Organization, Geneva Research on the immunology of parasitic infections has three outstanding characteristics: complexity, because successful parasitism involves the interweaving of i n t r i c a t e m e c h a n i s m s of host and p a r a s i t e ; fascinatzon in view of the m a n y insights into fundamental immunological, genetic and biochemical processes which study of this topic involves; and zmportance since parasitic infections constitute an enormous (although largely unappreciated) burden of disease on mankind, not to mention domestic and other animals, with social and economic consequences which it would be difficult to exaggerate. Although parasitic infections are attracting increasing attention from immunologists it must be confessed that knowledge is patchy and often inadequate as a basis for the development of vaccines and tests needed for disease control. For example, antigenic variation is considered to be one important way by which parasites may evade the host's immune response. Such variation is well-recognized in the surface coat protein of the trypanosomes which in Africa cause sleeping sickness in m a n and chronic disease in cattle. Knowledge of the size of the variant repertoire, of structural differences associated with variation, of the circumstances under which variation is induced and of the underlying genetic mechanisms is now rapidly emerging, aided recently by the use of monoclonal antibodies ~. Antigenic variation has also been demonstrated in one variety of malaria parasite, Plasmodium knowlesi, which infects monkeys. Red cells containing the schizonts of this parasite carry a series of different antigens recognizable by differential agglutinability 2, However, these potentially important antigens have not yet been characterized, nor is it agreed whether comparable variation exists in malarial parasites which infect man. There are a number of reasons for the present unsatisfactory state of knowledge. Immunologists only recently have established a general framework for the understanding of the immune response, including control and effector mechanisms. Prior to that it was © ElsevJer/North-llolland Biomedical Press 1980
hardly possible to begin to study the immunology of parasitic (or other) infections in a systematic way. It is encouraging to note how the study of immunoparasitology is now in turn feeding back information into the general body of immunology itself, for example information on the cytotoxic role of eosinophils and macrophages in combination with antibody which has been derived from work on immunity in schistosomiasis. Another important reason for the slow development of immunoparasitology is concerned with the antigenic heterogeneity of parasites. The newcomer to immunoparasitology may feel ill at ease when dealing with parasite antigens as compared with, for example, a clearly defined hapten-carrier system. Parasites present a great range of antigens to the host, and this often makes interpretation of experimental work difficult, and is a serious barrier to understanding the mechanisms of protective immunity. For example, antibodies to the merozoites of P. knowlesi have been shown to prevent the invasion of red cells in vztro~, but the relevant antigens have not been identified or characterized, nor is their relationship to the merozoite surface coat understood. Definition of parasite antigens is an essential and early step in the understanding of the immune response to parasites and the immunopathology of parasitic infections, and in the development of effective and safe vaccines. In the area of immunodiagnosis the absence of defined antigens causes endless frustration and confusion. Immunodiagnosis is, in general, a 'Cinderella' area of immunology; its practical importance is underrated by many immunologists, and its present state does not conform to the technical and conceptual development of immunology as a whole. This holds true not only for parasitic diseases but also for a large number of other conditions in which irnmunodiagnostic methods are in current use 4. At least so far as parasitic diseases go, much attention has been given to the developing of sensitive test systems for detecting a n t i g e n - a n t i b o d y reactions, e.g.
zmmzmologytoday,Auguft 1980 immunofluorescence, enzyme-linked immunosorbent assays and radioimmunoassays, but very little to the specificity of tests in terms of antigen. The reasons for this lack of attention to parasite antigens are not hard to find. Sometimes there may be a lack of understanding of the importance of antigen identification, but the major problem is the difficulty of obtaining adequate amounts of material to study from many of the important parasites. For example, the major sources of antigen for Plasmodium fa/ciparum malaria are infected h u m a n placentae and the smallscale in-vztro cultivation systems which have been so ably pioneered by Protessor Trager 5, both of which have serious limitations for formal immunochemical approaches. It is into this scene that the new technology of monoclonal antibodies has arrived.
Characteristics of monoclonal antibody technology There is an essential elegance and attraction in Kahler and Milstein's ~ idea of conferring cloning capability on individual antibody-producing lymphoid cells by fusion with myeloma cells. The homogeneous antibodies which are produced by such clones have come at an exceedingly opportune moment for the study of immunoparasitology, on account of the following characteristics. Spec~city. Antibodies produced by a single clone all have an identical combining site. Hence they show much greater epitope specificity than does the range of 'polyclonal' antibodies present in conventional antisera. A given monoclonal antibody may, of course, bind with different degrees of affinity to a set of different epitopes that has different degrees of complementarity to its combining site. Polyclonal antibodies, on the other hand, may react with many structurally unrelated epitopes, especially if complex mixtures of antigens have been used for immunization. Monoclonal antibodies can be used to bypass many of the difficult problems of antigen purification which arise when one works with small amounts of complex mixtures of parasite antigens. They will often be the reagents of choice for immunochemical isolation of individual antigens by affinity chromatography. T h e y are also valuable reagents for the study of antigenic heterogeneity of parasites in relation to such phenomena as stage specificity, antigenic variation and perhaps parasite genetics and taxonomy, although they have certain limitations in this respect which are mentioned below. Their great precision should enable a choice of antibody to be made which will avoid a common problem in immunodiagnostic methodology, that of cross-reactivity of antigens of different parasite species. Finally, and of very considerable importance, when a monoclonal antibody can be shown to have a specific biological activity, such as conferring resistance to infection, it both identifies the relevant antigen and also provides an immediate clue to the mechanism. An excellent example of this is provided by the
31 demonstration that a monoclonat antibody to Plasmodzum berghei sporozoites protects mice against sporozoite-induced infection 7. Uniformzty. Conventional antisera are notoriously difficult to standardize and no two antisera, even when obtained from the same animal, can be considered to be identical. The products of a single clone are, in contrast, uniform, so that for the first time the way is open to obtain reagents of uniform and definable specificity and affinity - - a matter of great importance, especially for immunodiagnostic work. In addition to their combining sites, monoclonal antibodies are uniform with respect to isotype and allotype. This makes them valuable tools for the study of the role of Ig classes and subclasses; this property has already been exploited in the study of schistosomiasis, as mentioned below. AvailabiliIy. The advantages of monoclonal specificity when sorting out antibody responses to complex antigenic mixtures have already been mentioned. The classical method of raising antibodies can now be supplemented by using infection itself as the stimulus to antibody production; for example monoclonal antibodies to schistosome antigens have been prepared successfully in Professor Capron's laboratory using spleen cells from infected animals ~. If antigen is prepared for immunization, the amount required is reduced to that necessary to immunize a few or even one mouse. Another aspect of the availability of monoclonal antibodies concerns the process of selection to obtain the appropriate clone. The products of many clones can be rapidly screened in a search tbr the required specificity. If cross-reactivity with antigens of another parasite species occurs frequently and is to be avoided, clones which produce such antibodies can be identified and discarded. Important, but antigenieally ' m i n o r ' antigens m a y also be identified by suitable screening. These are some of the benefits to be derived from the use of monoclonal antibodies. There are also some precautions to be observed in the use of these reagents. Monoclonat antibodies derive their specificity from their homogeneity, i.e. they react with one epitope only. Conventional antisera on the other hand usually contain antibodies that react with different epitopes of the same antigen molecule, as was first shown by Lapresle in his classic work on serum albumin. Unless more than one reacting epitope is present on the same molecule, monoclonal antibody cannot form a precipitin lattice; perhaps as a result of this the reaction kinetics are often slow. Monoclonal antibodies are fine tools for identifying molecular changes affecting the reacting epitope, but may fail to identify widespread changes elsewhere in the molecule, or the parasite. This has important consequences for the use of monoclonal antibodies in immunodiagnosis, 'for which it would seem desirable to use a battery of several monoclonal antibodies reacting with different epitopes, possibly on different molecules.
32 Another aspect of their fine specificity concerns the use of monoclonal antibodies for species or strain differentiation, such as may be required.to differentiate pathogenic from non-pathogenic trypanosomes or leishmania. Monoclonal antibodies may detect differences between parasites which are not necessarily relevant to the biological property in question. Perhaps the approach here will be to react panels of monoclonal antibodies and different parasites to find a pattern of monoclonal reactivity associated with the biological property in question. There are certain technical aspects of monoclonal antibody production which should also be considered. There may be chain deletion or other changes in secreted antibody, or a clone may stop secreting. One precaution is to preserve aliquots of important clones by freezing. To track down and clone promising antibody-producing hybrids needs a lot a work and requires careful planning, and the methods of antibody assay m a y be a limiting factor. Indirect immunofluorescence and radioimmunoassay have been widely used for work with parasite antibodies. Immunization schedules which include an intravenous injection of antigen three to four days before spleencell harvest a p p e a r to increase the yield of specific hybrids 9, and cloning efficiency may also be increased by the presence of peritoneal macrophages during cell culture. No doubt many other tricks will a p p e a r in future to increase cloning efficiency and to direct specificity towards wanted determinants. Sorting out the required cells would appear to be an ideal subject for automation. One other aspect of monoclonal antibody technology should be mentioned, which perhaps in the long run m a y prove to be its most important application. The difficulties inherent in obtaining antigens from m a n y parasites for experimental and immunodiagnostic purposes have already been indicated. These difficulties apply, a fortiori, to antigens for potential vaccines. New and cheap methods for antigen production are essential if vaccines are to be developed which are free from harmful material, and on a scale which has any relevance to the vast numbers of impoverished people at risk from major parasitic diseases. Genetic engineering through D N A hybridization m a y offer a solution. Vaccines may pass through a sequence of development, using monoclonal antibodies to define and isolate relevant antigens, genetic engineering for synthesis, and monoclonal antibodies again for monitoring and purification of the product. W h a t has b e e n d o n e so far
Some outstanding examples can be mentioned. In m a l a r i a , Professor R u t h N u s s e n z w e i g a n d her colleagues in New York have produced a monoclonal antibody to the protein coat of P. berghei sporozoites which reacts in the manner of antibodies which occur after sporozoite immunization 7. The reaction is detect-
immunologytoday, Augusl /980 able by immunofluorescence and the circumsporozoite precipitin test, and the monoclonal antibody confers protection on mice against sporozoite-induced infection. Dr L. Perrin and his colleagues in Geneva have produced monoclonal antibodies to the merozoites of P. falcz[mrum 1°. Of ten such antibodies which were shown to react against the surface of merozoites by immunofluorescence, two were found to inhibit the invasion of normal red blood cells by merozoites in vitro. The inhibition exceeded that observed using serum from repeatedly infected humans, and was as high as 98% after two cycles of cultivation. In a study of rodent malaria, R. R. Freeman and colleagues have shown that-monoclonal antibodies directed towards merozoite antigens but not other plasmodium antigens were protective in passive transfer experiments 1i. Extensive work on monoclonal antibodies to variant antigens of Trypanosoma brucei has been done by Dr T. Pearson and his collaborators in Nairobi. Eleven monoclonal antibodies showed specific reactivity with the immunizing variable coat antigen but did not cross-react with eight other variable antigens. Only a few of these antibodies bound to the surface of living trypanosomes carrying the a p p r o p r i a t e variable antigen, showing that some of the epitopes of these antigens are hidden on the surface of the living parasite (Pearson, T.W. and Kar, S.K., unpublished). These monoclonal antibodies have been used as immunoabsorbants for antigen preparation, and by combining two-dimensional gel electrophoresis with the use of monoclonaI antibody, analysis of antigens and antibodies was performed using very small amounts of material 12. Monoclonal antibodies to schistosome antigens have been produced by Professor Capron's group in Lille, using infected rats as a source of immune cells. These antibodies showed reactivity by immunofluorescence, especially with parasite membranes. Antibodies of IgE class showed a high titre of passive cutaneous anaphylactic activity and antibodies of the IgG2a subclass showed striking cytotoxic activity with eosinophils s. In conclusion, monoclonal antibody technology provides an extremely powerful new tool for the study of parasite immunology. These antibodies are of value in immunodiagnosis, in antigen preparation and analysis, and for biological studies of antibody activity. In view of the importance of the approach, two courses were recently c o n d u c t e d b y the U N D P / W o r l d B a n k / W H O Special Programme for Research and Training in Tropical Diseases to review the application and results of the technique as applied to parasites. Copies of the working papers of these courses which deal with theoretical and practical aspects of this technology are available on request from the Director of the Special Programme for Research and Training in Tropical Diseases, World Health Organization, Avenue Appia, Geneva.
zmmunology today, August 1980
33
1 am grateful to Dr V. Houba and Dr L. Perrin for help and advice in the preparation of this paper.
References 1 Pearson, T.W., Kar, S.K., McGaire, T.C. and Lundin, L.B. (1980) Proc. Natl. Acad. Sci. U.S.A. (in press) 2 Brown, K.N. (1974) in Parasites in lhe immumzedho.rl: mechamsms of survwal Ciba Foundation Symposium 2.5, p. 35, Associated Scientific Publishers,Amsterdam 3 Cohen, S. (1979) Proc. R. Soc. Ser. B 203,323 4 Friedman, H. (1979) Climcal Immunology NewsIezler October, p.6 5 Trager, W. andJensen, J.B. (1976) Science, 193, 673
reuiBws
6 K6hler, G. and Milstein, C. (1975) Nature (London) 256,495 7 Yoshida, N., Nussenzweig, R.S., Potoci~jak, P., Nussenzweig, V. and Aikawa, M. (1979) Sczence 207, 71 8 Verwaerde, C., Grzych, J.M., Bazin, H., Capron, M. and Capron, A. (1979) C. R. Acad. Sci. Ser. D 289, 725 9 St~ihli, C., Staehelin, T., Miggiano, V., Schmidt, J. and H~iring, P. (1980)J. Immunol. Methods 32, 297 10 Perrin, L.H., Ramirez, E., Erhfiang, Liu and Lambert, P.H. (1980) Chn. Exp. Immunol. (in press) 11 Freeman, R. R., Trejdosiewicz, A. J. and Cross, G. A. M. (1980) 2Vature(London) 284, 366 12 Pearson,T. and Anderson, L. (1980) Anal. Bzochem. 101, .377
1
H o w do immune response genes work? R. V. Blanden Microbiology Department, The John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra City, A.C.T. 2601, Australia. An inbred strain of experimental animal may give a strong or a weak i m m u n e response to a certain antigen. The genes determining this antigen-specific high or low responder status are termed immune response (b) genes, and in mice and guinea-pigs they m a p within the m a j o r h i s t o c o m p a t i b i l i t y gene complex ( M H C ) 1. There is a b u n d a n t evidence that Ir genes control the magnitude of responses mediated by thymus-derived (T) lymphocytes of various classes: cytotoxic T cells (Tc) ; T cells mediating delayed hypersensitivity (Td) ; helper T cells (Th); suppressor T cells (Ts). Cell-mediated responses can be affected directly by Ir genes that control the response of the effector cells (T c or Td), and indirectly by Ir genes controlling T h cells that amplify, or T s cells that suppress, the responses of effector cells. Antibody responses seem to be affected only by the indirect mechanisms (T h or Ts). - - The H - 2 gene c o m p l e x as a m o d e l Before proceeding to a discussion of possible mechanigms of Ir-gene action, it is essential to review briefly the relevant features of the M H C . The H - 2 gene complex of mice 2 is the most extensively studied example of M H C and will be used as the model system here, since it seems very likely that all other vertebrates with a well developed immune system possess a basically similar gene complex. The H - 2 complex consists of a set of genes on murine chromosome 17 that controls polymorphic cell surface antigens which provoke graft rejection. The known loci span sufficient DNA to code for about 2000 average proteins but at present much of the DNA has MHC
no defined function. The /i, D and L regions each contain one known locus coding for a polypeptide chain of a glycoprotein of mol. wt about 45,000. The I region (so named because it contains Ir genes) has loci coding for smaller polypeptides in glycoproteins of mol. wt 28,000 and 33,000. Antigenic specificity of these glycoproteins is determined by their amino-acid sequence, but the I and K regions also control glycolipids with antigenicity in the sugar moiety (Parish el al., unpublished) and therefore must either code for or control a set of glycosyl transferase enzymes.
T-cell recognition of antigenic patterns dependent upon self H-2 genes The H - 2 gene complex was discovered through Tcell-mediated allograft rejection a n d is being extensively studied using alloantibodies but in the present context its significance does not involve i m m u n e recognition of the cells of a foreign graft, but involves a form o f self r e c o g n i t i o n . The key point is that for the activation of To, T h or T d cells specific for any foreign antigen X (other than foreign M H C gene products and a few structurally similar molecules), it seems essential for self H - 2 genes to contribute to the total antigenic pattern which is recognized 3-s. The precise nature of these antigenic patterns or the T-cell receptors that recognize them is not yet known, but it is clear that a precursor T c cell is only activated when it recognizes an antigenic pattern which is dependent on both X and a K-, D-, or L-gene product being dis-. played on the same cell-surface membrane. Similarly, T h and T d cells respond to patterns dependent upon © Elsevier/North-Holland glomcdmal Press 1980
30
The role of monoclonal antibody technology in immunop ara sit ol ogy D. S. R o w e World Health Organization, Geneva Research on the immunology of parasitic infections has three outstanding characteristics: complexity, because successful parasitism involves the interweaving of i n t r i c a t e m e c h a n i s m s of host and p a r a s i t e ; fascinatzon in view of the m a n y insights into fundamental immunological, genetic and biochemical processes which study of this topic involves; and zmportance since parasitic infections constitute an enormous (although largely unappreciated) burden of disease on mankind, not to mention domestic and other animals, with social and economic consequences which it would be difficult to exaggerate. Although parasitic infections are attracting increasing attention from immunologists it must be confessed that knowledge is patchy and often inadequate as a basis for the development of vaccines and tests needed for disease control. For example, antigenic variation is considered to be one important way by which parasites may evade the host's immune response. Such variation is well-recognized in the surface coat protein of the trypanosomes which in Africa cause sleeping sickness in m a n and chronic disease in cattle. Knowledge of the size of the variant repertoire, of structural differences associated with variation, of the circumstances under which variation is induced and of the underlying genetic mechanisms is now rapidly emerging, aided recently by the use of monoclonal antibodies ~. Antigenic variation has also been demonstrated in one variety of malaria parasite, Plasmodium knowlesi, which infects monkeys. Red cells containing the schizonts of this parasite carry a series of different antigens recognizable by differential agglutinability 2, However, these potentially important antigens have not yet been characterized, nor is it agreed whether comparable variation exists in malarial parasites which infect man. There are a number of reasons for the present unsatisfactory state of knowledge. Immunologists only recently have established a general framework for the understanding of the immune response, including control and effector mechanisms. Prior to that it was © ElsevJer/North-llolland Biomedical Press 1980
hardly possible to begin to study the immunology of parasitic (or other) infections in a systematic way. It is encouraging to note how the study of immunoparasitology is now in turn feeding back information into the general body of immunology itself, for example information on the cytotoxic role of eosinophils and macrophages in combination with antibody which has been derived from work on immunity in schistosomiasis. Another important reason for the slow development of immunoparasitology is concerned with the antigenic heterogeneity of parasites. The newcomer to immunoparasitology may feel ill at ease when dealing with parasite antigens as compared with, for example, a clearly defined hapten-carrier system. Parasites present a great range of antigens to the host, and this often makes interpretation of experimental work difficult, and is a serious barrier to understanding the mechanisms of protective immunity. For example, antibodies to the merozoites of P. knowlesi have been shown to prevent the invasion of red cells in vztro~, but the relevant antigens have not been identified or characterized, nor is their relationship to the merozoite surface coat understood. Definition of parasite antigens is an essential and early step in the understanding of the immune response to parasites and the immunopathology of parasitic infections, and in the development of effective and safe vaccines. In the area of immunodiagnosis the absence of defined antigens causes endless frustration and confusion. Immunodiagnosis is, in general, a 'Cinderella' area of immunology; its practical importance is underrated by many immunologists, and its present state does not conform to the technical and conceptual development of immunology as a whole. This holds true not only for parasitic diseases but also for a large number of other conditions in which irnmunodiagnostic methods are in current use 4. At least so far as parasitic diseases go, much attention has been given to the developing of sensitive test systems for detecting a n t i g e n - a n t i b o d y reactions, e.g.
zmmzmologytoday,Auguft 1980 immunofluorescence, enzyme-linked immunosorbent assays and radioimmunoassays, but very little to the specificity of tests in terms of antigen. The reasons for this lack of attention to parasite antigens are not hard to find. Sometimes there may be a lack of understanding of the importance of antigen identification, but the major problem is the difficulty of obtaining adequate amounts of material to study from many of the important parasites. For example, the major sources of antigen for Plasmodium fa/ciparum malaria are infected h u m a n placentae and the smallscale in-vztro cultivation systems which have been so ably pioneered by Protessor Trager 5, both of which have serious limitations for formal immunochemical approaches. It is into this scene that the new technology of monoclonal antibodies has arrived.
Characteristics of monoclonal antibody technology There is an essential elegance and attraction in Kahler and Milstein's ~ idea of conferring cloning capability on individual antibody-producing lymphoid cells by fusion with myeloma cells. The homogeneous antibodies which are produced by such clones have come at an exceedingly opportune moment for the study of immunoparasitology, on account of the following characteristics. Spec~city. Antibodies produced by a single clone all have an identical combining site. Hence they show much greater epitope specificity than does the range of 'polyclonal' antibodies present in conventional antisera. A given monoclonal antibody may, of course, bind with different degrees of affinity to a set of different epitopes that has different degrees of complementarity to its combining site. Polyclonal antibodies, on the other hand, may react with many structurally unrelated epitopes, especially if complex mixtures of antigens have been used for immunization. Monoclonal antibodies can be used to bypass many of the difficult problems of antigen purification which arise when one works with small amounts of complex mixtures of parasite antigens. They will often be the reagents of choice for immunochemical isolation of individual antigens by affinity chromatography. T h e y are also valuable reagents for the study of antigenic heterogeneity of parasites in relation to such phenomena as stage specificity, antigenic variation and perhaps parasite genetics and taxonomy, although they have certain limitations in this respect which are mentioned below. Their great precision should enable a choice of antibody to be made which will avoid a common problem in immunodiagnostic methodology, that of cross-reactivity of antigens of different parasite species. Finally, and of very considerable importance, when a monoclonal antibody can be shown to have a specific biological activity, such as conferring resistance to infection, it both identifies the relevant antigen and also provides an immediate clue to the mechanism. An excellent example of this is provided by the
31 demonstration that a monoclonat antibody to Plasmodzum berghei sporozoites protects mice against sporozoite-induced infection 7. Uniformzty. Conventional antisera are notoriously difficult to standardize and no two antisera, even when obtained from the same animal, can be considered to be identical. The products of a single clone are, in contrast, uniform, so that for the first time the way is open to obtain reagents of uniform and definable specificity and affinity - - a matter of great importance, especially for immunodiagnostic work. In addition to their combining sites, monoclonal antibodies are uniform with respect to isotype and allotype. This makes them valuable tools for the study of the role of Ig classes and subclasses; this property has already been exploited in the study of schistosomiasis, as mentioned below. AvailabiliIy. The advantages of monoclonal specificity when sorting out antibody responses to complex antigenic mixtures have already been mentioned. The classical method of raising antibodies can now be supplemented by using infection itself as the stimulus to antibody production; for example monoclonal antibodies to schistosome antigens have been prepared successfully in Professor Capron's laboratory using spleen cells from infected animals ~. If antigen is prepared for immunization, the amount required is reduced to that necessary to immunize a few or even one mouse. Another aspect of the availability of monoclonal antibodies concerns the process of selection to obtain the appropriate clone. The products of many clones can be rapidly screened in a search tbr the required specificity. If cross-reactivity with antigens of another parasite species occurs frequently and is to be avoided, clones which produce such antibodies can be identified and discarded. Important, but antigenieally ' m i n o r ' antigens m a y also be identified by suitable screening. These are some of the benefits to be derived from the use of monoclonal antibodies. There are also some precautions to be observed in the use of these reagents. Monoclonat antibodies derive their specificity from their homogeneity, i.e. they react with one epitope only. Conventional antisera on the other hand usually contain antibodies that react with different epitopes of the same antigen molecule, as was first shown by Lapresle in his classic work on serum albumin. Unless more than one reacting epitope is present on the same molecule, monoclonal antibody cannot form a precipitin lattice; perhaps as a result of this the reaction kinetics are often slow. Monoclonal antibodies are fine tools for identifying molecular changes affecting the reacting epitope, but may fail to identify widespread changes elsewhere in the molecule, or the parasite. This has important consequences for the use of monoclonal antibodies in immunodiagnosis, 'for which it would seem desirable to use a battery of several monoclonal antibodies reacting with different epitopes, possibly on different molecules.
32 Another aspect of their fine specificity concerns the use of monoclonal antibodies for species or strain differentiation, such as may be required.to differentiate pathogenic from non-pathogenic trypanosomes or leishmania. Monoclonal antibodies may detect differences between parasites which are not necessarily relevant to the biological property in question. Perhaps the approach here will be to react panels of monoclonal antibodies and different parasites to find a pattern of monoclonal reactivity associated with the biological property in question. There are certain technical aspects of monoclonal antibody production which should also be considered. There may be chain deletion or other changes in secreted antibody, or a clone may stop secreting. One precaution is to preserve aliquots of important clones by freezing. To track down and clone promising antibody-producing hybrids needs a lot a work and requires careful planning, and the methods of antibody assay m a y be a limiting factor. Indirect immunofluorescence and radioimmunoassay have been widely used for work with parasite antibodies. Immunization schedules which include an intravenous injection of antigen three to four days before spleencell harvest a p p e a r to increase the yield of specific hybrids 9, and cloning efficiency may also be increased by the presence of peritoneal macrophages during cell culture. No doubt many other tricks will a p p e a r in future to increase cloning efficiency and to direct specificity towards wanted determinants. Sorting out the required cells would appear to be an ideal subject for automation. One other aspect of monoclonal antibody technology should be mentioned, which perhaps in the long run m a y prove to be its most important application. The difficulties inherent in obtaining antigens from m a n y parasites for experimental and immunodiagnostic purposes have already been indicated. These difficulties apply, a fortiori, to antigens for potential vaccines. New and cheap methods for antigen production are essential if vaccines are to be developed which are free from harmful material, and on a scale which has any relevance to the vast numbers of impoverished people at risk from major parasitic diseases. Genetic engineering through D N A hybridization m a y offer a solution. Vaccines may pass through a sequence of development, using monoclonal antibodies to define and isolate relevant antigens, genetic engineering for synthesis, and monoclonal antibodies again for monitoring and purification of the product. W h a t has b e e n d o n e so far
Some outstanding examples can be mentioned. In m a l a r i a , Professor R u t h N u s s e n z w e i g a n d her colleagues in New York have produced a monoclonal antibody to the protein coat of P. berghei sporozoites which reacts in the manner of antibodies which occur after sporozoite immunization 7. The reaction is detect-
immunologytoday, Augusl /980 able by immunofluorescence and the circumsporozoite precipitin test, and the monoclonal antibody confers protection on mice against sporozoite-induced infection. Dr L. Perrin and his colleagues in Geneva have produced monoclonal antibodies to the merozoites of P. falcz[mrum 1°. Of ten such antibodies which were shown to react against the surface of merozoites by immunofluorescence, two were found to inhibit the invasion of normal red blood cells by merozoites in vitro. The inhibition exceeded that observed using serum from repeatedly infected humans, and was as high as 98% after two cycles of cultivation. In a study of rodent malaria, R. R. Freeman and colleagues have shown that-monoclonal antibodies directed towards merozoite antigens but not other plasmodium antigens were protective in passive transfer experiments 1i. Extensive work on monoclonal antibodies to variant antigens of Trypanosoma brucei has been done by Dr T. Pearson and his collaborators in Nairobi. Eleven monoclonal antibodies showed specific reactivity with the immunizing variable coat antigen but did not cross-react with eight other variable antigens. Only a few of these antibodies bound to the surface of living trypanosomes carrying the a p p r o p r i a t e variable antigen, showing that some of the epitopes of these antigens are hidden on the surface of the living parasite (Pearson, T.W. and Kar, S.K., unpublished). These monoclonal antibodies have been used as immunoabsorbants for antigen preparation, and by combining two-dimensional gel electrophoresis with the use of monoclonaI antibody, analysis of antigens and antibodies was performed using very small amounts of material 12. Monoclonal antibodies to schistosome antigens have been produced by Professor Capron's group in Lille, using infected rats as a source of immune cells. These antibodies showed reactivity by immunofluorescence, especially with parasite membranes. Antibodies of IgE class showed a high titre of passive cutaneous anaphylactic activity and antibodies of the IgG2a subclass showed striking cytotoxic activity with eosinophils s. In conclusion, monoclonal antibody technology provides an extremely powerful new tool for the study of parasite immunology. These antibodies are of value in immunodiagnosis, in antigen preparation and analysis, and for biological studies of antibody activity. In view of the importance of the approach, two courses were recently c o n d u c t e d b y the U N D P / W o r l d B a n k / W H O Special Programme for Research and Training in Tropical Diseases to review the application and results of the technique as applied to parasites. Copies of the working papers of these courses which deal with theoretical and practical aspects of this technology are available on request from the Director of the Special Programme for Research and Training in Tropical Diseases, World Health Organization, Avenue Appia, Geneva.
zmmunology today, August 1980
33
1 am grateful to Dr V. Houba and Dr L. Perrin for help and advice in the preparation of this paper.
References 1 Pearson, T.W., Kar, S.K., McGaire, T.C. and Lundin, L.B. (1980) Proc. Natl. Acad. Sci. U.S.A. (in press) 2 Brown, K.N. (1974) in Parasites in lhe immumzedho.rl: mechamsms of survwal Ciba Foundation Symposium 2.5, p. 35, Associated Scientific Publishers,Amsterdam 3 Cohen, S. (1979) Proc. R. Soc. Ser. B 203,323 4 Friedman, H. (1979) Climcal Immunology NewsIezler October, p.6 5 Trager, W. andJensen, J.B. (1976) Science, 193, 673
reuiBws
6 K6hler, G. and Milstein, C. (1975) Nature (London) 256,495 7 Yoshida, N., Nussenzweig, R.S., Potoci~jak, P., Nussenzweig, V. and Aikawa, M. (1979) Sczence 207, 71 8 Verwaerde, C., Grzych, J.M., Bazin, H., Capron, M. and Capron, A. (1979) C. R. Acad. Sci. Ser. D 289, 725 9 St~ihli, C., Staehelin, T., Miggiano, V., Schmidt, J. and H~iring, P. (1980)J. Immunol. Methods 32, 297 10 Perrin, L.H., Ramirez, E., Erhfiang, Liu and Lambert, P.H. (1980) Chn. Exp. Immunol. (in press) 11 Freeman, R. R., Trejdosiewicz, A. J. and Cross, G. A. M. (1980) 2Vature(London) 284, 366 12 Pearson,T. and Anderson, L. (1980) Anal. Bzochem. 101, .377
1
H o w do immune response genes work? R. V. Blanden Microbiology Department, The John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra City, A.C.T. 2601, Australia. An inbred strain of experimental animal may give a strong or a weak i m m u n e response to a certain antigen. The genes determining this antigen-specific high or low responder status are termed immune response (b) genes, and in mice and guinea-pigs they m a p within the m a j o r h i s t o c o m p a t i b i l i t y gene complex ( M H C ) 1. There is a b u n d a n t evidence that Ir genes control the magnitude of responses mediated by thymus-derived (T) lymphocytes of various classes: cytotoxic T cells (Tc) ; T cells mediating delayed hypersensitivity (Td) ; helper T cells (Th); suppressor T cells (Ts). Cell-mediated responses can be affected directly by Ir genes that control the response of the effector cells (T c or Td), and indirectly by Ir genes controlling T h cells that amplify, or T s cells that suppress, the responses of effector cells. Antibody responses seem to be affected only by the indirect mechanisms (T h or Ts). - - The H - 2 gene c o m p l e x as a m o d e l Before proceeding to a discussion of possible mechanigms of Ir-gene action, it is essential to review briefly the relevant features of the M H C . The H - 2 gene complex of mice 2 is the most extensively studied example of M H C and will be used as the model system here, since it seems very likely that all other vertebrates with a well developed immune system possess a basically similar gene complex. The H - 2 complex consists of a set of genes on murine chromosome 17 that controls polymorphic cell surface antigens which provoke graft rejection. The known loci span sufficient DNA to code for about 2000 average proteins but at present much of the DNA has MHC
no defined function. The /i, D and L regions each contain one known locus coding for a polypeptide chain of a glycoprotein of mol. wt about 45,000. The I region (so named because it contains Ir genes) has loci coding for smaller polypeptides in glycoproteins of mol. wt 28,000 and 33,000. Antigenic specificity of these glycoproteins is determined by their amino-acid sequence, but the I and K regions also control glycolipids with antigenicity in the sugar moiety (Parish el al., unpublished) and therefore must either code for or control a set of glycosyl transferase enzymes.
T-cell recognition of antigenic patterns dependent upon self H-2 genes The H - 2 gene complex was discovered through Tcell-mediated allograft rejection a n d is being extensively studied using alloantibodies but in the present context its significance does not involve i m m u n e recognition of the cells of a foreign graft, but involves a form o f self r e c o g n i t i o n . The key point is that for the activation of To, T h or T d cells specific for any foreign antigen X (other than foreign M H C gene products and a few structurally similar molecules), it seems essential for self H - 2 genes to contribute to the total antigenic pattern which is recognized 3-s. The precise nature of these antigenic patterns or the T-cell receptors that recognize them is not yet known, but it is clear that a precursor T c cell is only activated when it recognizes an antigenic pattern which is dependent on both X and a K-, D-, or L-gene product being dis-. played on the same cell-surface membrane. Similarly, T h and T d cells respond to patterns dependent upon © Elsevier/North-Holland glomcdmal Press 1980
